Nano-flow liquid chromatographic apparatus having robust capillary tubing

ABSTRACT

A chemical processing apparatus includes a separation column, a pump unit configured to support nano-flow processing, at least one fluid transport tube for transporting the fluid between the pump unit and the separation column, and a connector disposed adjacent to an inlet or outlet end of the at least one transport tube. The fluid transport tube and/or the separation column includes an outer tube of a metallic material, a fused-silica capillary disposed in the outer tube, and an intermediate tube including a polymeric material disposed between and bonded to both the outer tube and the capillary.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalPatent Application No. 60/719,070, filed on Sep. 21, 2005, the entirecontents of which is incorporated herein by reference.

TECHNICAL FIELD

The invention generally relates to chromatography instruments that haveplumbing, including tubing and connectors, designed to accommodate highpressure and/or low flow rates.

BACKGROUND INFORMATION

Various instruments utilize conduits for transportation of processfluids and sample compounds and/or for separation of sample compounds.For example, chemical-analysis instruments that utilize liquidchromatography (LC), capillary electrophoresis (CE) or capillaryelectro-chromatography (CEC) perform separation of sample compounds asthe sample passes through a column. Such instruments include plumbing,such as conduits and connectors, that transport a variety of materials,such as solvents and sample compounds.

In addition to tubing, used, for example, for separation column(s)and/or plumbing, liquid-chromatography instruments typically includereservoirs, pumps, filters, check valves, sample-injection valves, andsample compound detectors. Typically, solvents are stored in reservoirsand delivered as required via reciprocating-cylinder based pumps. Samplematerials are often injected via syringe-type pumps.

In some cases, separation columns include one or more electrodes topermit application of a voltage to a sample-containing fluid passingthrough and/or exiting from the conduit. CEC, for example, utilizes anelectro-osmotic flow (EOF) to propel a mobile phase through achromatographic column. In contrast, liquid chromatography, such ashigh-performance liquid chromatography (HPLC), relies on pressure topropel a fluid through a column.

Suitable analytical-instrument tubing withstands pressures encounteringduring fabrication and use, is reliable through repeated use, and hasphysical and chemical compatibility with process and sample compounds.Generally, a tubing material should not corrode or leach, and samplecompounds should not adhere to the tube (unless required for aseparation process.)

For HPLC and higher-pressure applications, tubing is typically made fromstainless steel or fused silica to provide suitable strength andcleanliness. Such tubing is typically joined to other components viastainless steel connectors.

Stainless steel, however, has disadvantages in some applications due toits biocompatibility limits in comparison to some other materials; someorganic molecules tend to adhere to the inner walls of steel tubing, andcomponents of a steel alloy at times leach into fluid passing throughthe tubing. Organic molecules generally are less likely to stick tofused silica or suitable polymeric materials than to steel. Fused silicatubing, however, is vulnerable to fracturing while polymeric materialsgenerally have relatively poor strength.

Typically, tubing must also be compatible with connectors that providefluidic connections to other components of an instrument. Problemsassociated with the design and use of connector fittings areparticularly difficult for high-pressure fabrication and operation. Forexample, pressures in the range of 1,000-5,000 pounds per square inch(psi) or higher are often utilized in liquid chromatography, and must beaccommodated without undesirable amounts of leakage.

SUMMARY OF THE INVENTION

The invention arises, in part, from the realization that a nano-flow LCapparatus advantageously includes fluid transport tubing configured witha fused silica inner tube having a narrow inner diameter, a steel outertube, and a polymer intermediate tube, as well as high-pressureconnectors configured to mate with the steel outer tube. Conventionalconnectors are optionally used in such an apparatus.

The layered tubing provides the narrow dimensions and other benefits offused silica capillary plumbing in a nano-flow LC system, while alsoproviding the mechanical stability and good connector interface of steeltubing in high pressure applications. The steel tubing protects thecapillary from damage from connectors, and the intermediate tube fixesthe position of the capillary relative to the steel tubing so that thecapillary does not move in response to pressure transmitted by apressurized fluid.

Apparatus of the inventions solve problems of low efficiency, distortedpeak shape, and/or leaking fittings of some prior nano-flow LCapparatus. Various nano-flow apparatus of the invention include, forexample, layered tubing connected to a separation column, a sampleinjector, and/or a detector.

Accordingly, one embodiment of the invention features a chemicalprocessing apparatus. The apparatus includes a separation column, a pumpunit, at least one fluid transport tube for transporting the fluidbetween the pump unit and the separation column, and a connectordisposed adjacent to an inlet or outlet end of the at least onetransport tube.

The connector includes, for example, a ferrule, a compression screw, anda fitting that receives the compression screw. The connector provides asubstantially fluid-tight connection between the end of the at least onetransport tube and an output port of the pump unit or an input port ofthe column.

The pump unit is configured to deliver, to the separation column, afluid at a pressure of at least about 10,000 psi at a flow rate of 100μL/min or lower.

The fluid transport tube includes an outer tube including a metallicmaterial, a liner tube including fused silica disposed in the outertube, and an intermediate tube including a polymeric material disposedbetween and bonded to both the outer tube and the liner tube.

Another embodiment of the invention features LC tubing suitable foroperation at pressures up to about 10,000 psi to 15,000 psi or greaterand providing relatively good biocompatibility. The tubing optionally isfabricated by inserting a polymeric tube into a high-strength outersteel tube, inserting a silica capillary into the polymeric tube, andmelt bonding the polymeric tube to the outer tube and the capillary.

In some alternative embodiments, a portion of a polymeric tube is meltedto form a bond with adjacent tubes. The bond inhibits sliding movementof, for example, an inner capillary relative to an outer tube and/orprovides a leakage barrier for the interfaces with the inner and/orouter tubes.

Some embodiments of such tubing have a variety of advantages over someconventional tubing. For example, some embodiments are relatively easyand inexpensive to manufacture. Some embodiments are compatible withcommonly available metallic-based high-pressure connectors. Some ofthese embodiments are fabricated from standard stainless steel ortitanium tubing that is suitable for operation at relatively highpressures.

Thus, as one example, a relatively high-pressure and low flow-ratecompatible conduit is constructed at a relatively low cost from readilyavailable components and integrated with other components of a nano-flowinstrument by utilizing standard high-pressure connectors.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference characters generally refer to the sameparts throughout the different views. Also, the drawings are notnecessarily to scale, emphasis instead generally being placed uponillustrating the principles of the invention.

FIG. 1 is a flow diagram of a method for fabricatinganalytical-instrument tubing, in accordance with one embodiment of theinvention;

FIG. 2 a is a cross-sectional diagram of tubing at an intermediate stageof fabrication, in accordance with one embodiment of the invention;

FIG. 2 b is a cross-sectional diagram of the tubing of FIG. 2 a at alater stage of fabrication;

FIG. 3 is an angled end view of a tube, in accordance with oneembodiment of the invention;

FIG. 4 is a cross-sectional diagram of a portion of a connector and atube, in accordance with one embodiment of the invention; and

FIG. 5 is a block diagram of an analytical instrument, in accordancewith one embodiment of the invention.

DESCRIPTION

The phrases “chromatographic system,” “chromatographic module,”“chromatographic instrument,” and the like herein refer to equipmentused to perform chemical separations. Such equipment is a portion of aninstrument that includes other components or is a standalone unit.Chromatographic equipment typically moves fluids under pressure and/orelectrical forces.

Depending on context, the description provided herein of someillustrative embodiments of the invention interchangeably uses the words“tube,” “conduit,” and/or “pipe.” Depending on context, the word“capillary” refers to fused-silica tubes and/or refers to tubes having arelatively narrow inner diameter. Tubes define an interior passageway,herein also referred to interchangeably as a lumen, bore, or channel.The word “column” herein refers to a tube that is used for separation ofcompounds in a sample, or is used to propel fluids in an electrokineticpump.

The word “biocompatiblity” herein relates to the tendency of someorganic materials to adhere to a particular tube material, as would beunderstood by one of ordinary skill. For example, fused silica isgenerally considered to be more biocompatible than is steel becauseorganic molecules are typically less likely to adhere to fused silicathan to a steel alloy.

The terms “nano-flow” and “nanoflow” are used herein to refer to fluidflow rates of less than about 100 μL/min. Nano-flow rates are useful,for example, in some applications of chromatography performed atpressures of 1,000 psi or greater, and at even higher pressures, such as10,000 psi or greater.

Some embodiments of the invention involve apparatus that include bothchromatographic and mass-spectrometric components. In some of theseembodiments, a chromatographic component is placed in fluidcommunication with a mass-spectrometric component through use of anappropriate interface, such as an electrospray-ionization interface, asknown to one of ordinary skill. Some appropriate interfaces at timescreate or maintain separated materials in an ionic form and typicallyplace a stream of fluid containing the ions into an atmosphere where thestream is vaporized and the ions are received in an orifice formass-spectrometric analyses.

FIG. 1 is a flow diagram that illustrates a method 100 for fabricatingtubing for use in a chemical-processing apparatus, such as a HPLCapparatus, in accordance with one embodiment of the invention. Themethod 100 includes providing (Step 110) an inner tube that is formed atleast in part from a polymeric material, providing (Step 120) an outertube that is formed at least in part from a material having a greateryield strength than the polymeric material, inserting (Step 130) theinner tube into the outer tube, and bonding (Step 140) the inner tube tothe outer tube by melting at least a portion of the polymeric material.Upon solidification of the melted portion, a fixed contact is formedbetween the inner and outer tubes.

Some alternative embodiments, described in more detail below withreference to FIG. 3, include a liner tube formed from fused silica,which is disposed in the polymeric tube; in such embodiments thepolymeric tube is referred to as an “intermediate” tube.

Now also referring to FIGS. 2 a and FIG. 2 b, the method 100 optionallyincludes extracting (Step 145) heat from an interior surface of theinner tube during melting, and/or includes trimming (Step 150) one ormore portions of the inner tube after bonding the inner tube to theouter tube. FIGS. 2 a and FIG. 2 b illustrate cross-sectional views of atube 200 as it appears during heat extraction (Step 145) and after beingtrimmed (Step 150), in accordance with one alternative implementation ofthe method 100.

The tube 200 includes an outer tube 210 and an inner tube 220. Asdescribed in more detail below, the outer tube 210 is formed of amaterial that provides suitable strength and reliability while the innertube 220 is formed of a material that provides melt-bonding capabilityand/or suitable biocompatibility. Upon completion of fabrication, thetube 200 is suitable for use as, for example, a transport conduit orcolumn in a chromatographic system.

As illustrated in FIG. 2 a, optionally, the inner tube 220 is initiallyselected to have a greater length than the length of the outer tube 210.In some embodiments of the method 100, the inner tube shrinks in lengthduring bonding. Hence, selection of an inner tube 220 having a greaterlength in some cases avoids shrinkage of the inner tube 220 to a lengthless than that of the outer tube 220.

Subsequent to bonding of the inner tube 220 to the outer tube 220, ifdesired, the inner tube is trimmed (Step 150). In the illustratedexample, the inner tube 220 is trimmed flush with the outer tube 210. Inalternative embodiments of the invention, inner and/or outer tubes aretrimmed and/or otherwise shaped as desired for compatibility with othercomponents of an analytical system. Trimming (Step 150) in support ofcompatibility with conduit connectors is described below, in part withreference to FIG. 4.

The inner tube 220 defines a lumen through which material—such assolvent and/or sample material—flows. As described in more detail below,the outer tube 210 provides, in part, mechanical support while the innertube 220 provides, in part, compatibility with a material flowingthrough the tube.

In various embodiments, the polymeric material is selected for itsability to form a melt bond to the outer tube and/or for itsbiocompatibility. For example, biocompatibility with proteins andpeptides is important in some applications. In some embodiments, theinner tube is at least partially formed of any suitable meltablepolymer, including known thermoplastic polymers.

The polyaryl-ether-ketones, for example, provide one class ofthermoplastic polymers that also has good biocompatibility. One of thesuitable polymeric materials of this class is polyether-ether-ketone,such as PEEK polymer (available from Victrex PLC, Lancashire, UnitedKingdom.)

Some embodiments utilize other polymers, for example, fluoropolymerssuch as polytetrafluorothylene (available as TEFLON polymer from DupontEngineering Polymers, Newark, Delware), chlorotetrafluoroethylene,polychlorotrifluoroethylene (available as NEOFLON PCTFE fluoropolymerfrom Fluorotherm Polymers, Inc., Fairfield, N.J.), and modifiedcopolymer fluoropolymers (for example, a modified copolymer oftetrafluoroethylene and ethylene available as DUPONT TEFZELfluoropolymer, which is resistant to concentrated nitric acid orsulfuric acid), and other polymers, such as polyimide (available asDUPONT VESPEL polyimide.)

In some embodiments, the inner tube is formed of a composite material.For example, in some of these embodiments, the inner tub is formed of amixture of a polymer, such as polyether-ether-ketone, and about 5% byweight of glass, fiberglass, carbon, and/or or other particles and/orfibers.

The material of the outer tube is selected from any suitable materials,including known materials, to provide, for example, a sufficient levelof mechanical strength to support fabrication and/or operatingconditions. In one embodiment, a desired level of mechanical strength isobtained by the combination of an outer tube(s) and an inner tube(s).For example, the materials and wall thicknesses of the inner and outertubes are selected to perform HPLC (at, for example, about 2 kpsi toabout 5 kpsi,) or to operate at higher pressures up to about 10 kpsi to15 kpsi or higher.

Steel and titanium, for example, have relatively high yield strength,and are thus suitable for high-pressure operation of a transport tubing,column tubing, etc. For outer tubing, some embodiments utilize standardtubing known to those having ordinary skill in the high-pressurechromatographic arts. One suitable standard tubing is 1/16 inch outerdiameter (OD) 316 alloy stainless steel tubing. The inner diameter (ID)of the steel tubing is selected as desired from, for example, standardavailable IDs. Standard IDs are available as small as about 4 mil (about100 μm.)

In some embodiments, an OD of an inner tube is selected to provide aslidable fit within the selected outer tubing. An ID of an inner tube isselected as desired. For example, an ID can be selected to be as smallas about 2 mil (about 50 μm) or less.

After inserting (Step 130) the inner tube, bonding is initiated byheating (Step 140) sufficiently to melt at least a portion of the innertube adjacent to the inner surface of the outer tube. Upon cooling, themelted portion solidifies and forms a fixed contact between the innerand outer tubes.

The inner tube is heated in any suitable manner. In one embodiment, theinner tube is heated indirectly by heating an adjacent portion of theouter tube. For example, the inner tube is heated by heating the outertube sufficiently to raise the temperature of portions of the inner tubeto at least a melting point temperature.

For example, in some embodiments, the entire outer tube is heated,uniformly or non-uniformly. In other embodiments, heat is directed onlyto one or more portions of the outer tube. As illustrated in FIG. 2 a.,in one embodiment, heat is directed to end portions of the outer tube210. In one alternative of this embodiment, two bonded regions areformed to restrict movement of the inner tube 220 within the outer tube210 and to restrict leakage of fluid past the bonded regions into thenon-bonded interfacial space between the inner and out tubes 210, 220.

Heat is directed at the outer tube in any suitable manner, includingknown heating methods. For example, the inner and outer tubes, orportions of the tubes, are placed in one or more ovens or in cavities ofheatable blocks of aluminum or steel. Such blocks are heated by, forexample, resistive heaters or a heated platten. Other options forheating, such as induction heating, are available and any suitablemethod may be used. Various embodiments utilize any method of heattransfer that provides the desired bonding temperature and environment.

The portion of the inner tube that is melted (Step 140) has itstemperature profile controlled as desired. For example, the temperatureis raised gradually to a desired temperature over a period of seconds orminutes or hours. Alternatively, the portion of the inner tube is meltednearly instantaneously. In some embodiments, a suitable temperatureprofile that supports a good bond is empirically or theoreticallydetermined.

In some embodiments, heating over a period of several minutes is helpfulto obtain a good bond. It is desirable in some cases to controllablyheat and melt the portion of the inner tube to obtain repeatable resultsand to avoid incorporation of bubbles or voids within a bonded region.

In some embodiments, it is undesirable to overheat the polymericmaterial of the inner tube when thermal breakdown or decomposition ispossible. One embodiment utilizes a non-oxidizing atmosphere duringheating.

After heating, the inner and outer tubes are either passively oractively cooled to ambient temperature. Cooling is accelerated by, forexample, any suitable method that maintains the chemical and structuralintegrity of the bond and components.

Some alternative implementations of extracting (Step 145) heat duringmelting (Step 140) are now described. To extract heat, a fluid, such asa gas or liquid, is directed through a lumen defined by the innermosttube. In some embodiments, the fluid is a substantially inert gas, suchas nitrogen or argon.

The fluid is used, for example, to ensure that melting remains localizedand does not extend to the inner surface of the polymeric-material tube.The fluid is thus used, in some cases, to maintain a passageway throughthe inner tube during melting (Step 140).

In one embodiment, the flow of a gas through the tube is controlled bymonitoring the pressure drop of the gas across the tube (i.e., thedifference in pressure between an inlet end and an outlet end of thetube.) Desirable pressure drops are, for example, in a range of about 10psi to about 100 psi. An increase in the selected pressure drop is oftendesirable for greater lengths of tubing and/or for smaller diameters ofa passageway.

A suitable pressure drop is determined, for example, empirically. Forparticular selected materials and tube dimensions, a suitable pressureis determined at which the passageway through the tube remains openduring bonding.

In one embodiment, gas is directed into the tube at one end of the tubewhile a portion of the tube adjacent to the opposite end of the tube isheated to form a bond adjacent to that end. Gas is then directed intothe bonded end of the tube, and the now opposite end is heated to form abond adjacent to that end. In this manner, a passageway is maintainedthrough a lumen having an ID of as small as about 50 μm or less.

The remaining description, below, is directed primarily to someembodiments that utilize a steel outer tube and a polyether-ether-ketoneinner tube. One having ordinary skill will understand, however, thatprinciples of the invention are applicable to a broader range ofmaterials and processing conditions.

Melting (Step 140), in one illustrative case, is obtained by heatingportions of the inner tube to a temperature somewhat above the meltingpoint temperature. In one embodiment, for example, thepolyether-ether-ketone portion is heated to a temperature of betweenabout 385° C. to about 405° C. The polymer is heated at the desiredtemperature for a period of time of about 1 to about 3 minutes, althoughthe invention is not limited to such. It is often desirable to heatneighboring portions of the inner and outer tubes to a similar or sametemperature during melting (Step 140) to obtain a good bond between theinner and outer tubes.

In one illustrative embodiment, an analytical-instrument tube includesan inner tube and an outer tube of the following dimensions andcomposition. The outer tube is formed of drawn 316 stainless steel andthe inner tube is formed of extruded polyether-ether-ketone. The innertube has an inner diameter (ID) of 2 mil (50 μm) or 2.5 mil (60 μm). Theouter tube has an outer diameter of 1/16 inch, and has an ID selected tobe compatible with the OD of the inner tube. The word “compatible” isherein used to mean that the inner tube can be inserted into the outertube. Preferably, during insertion, the inner tube is not damaged andthere is some contact around the circumference of the inner tube, i.e.,there is a minimal gap between the inner and outer tubes. One havingordinary skill will understand this example is merely illustrative andnon-limiting.

Optionally, more than one inner tube and/or more than one outer tube areutilized to fabricate tubing. For example, some embodiments entailfabrication of a conduit including two or more outer tubes disposed in arow (along the conduit) and/or disposed within one another. For example,in one embodiment, multiple inner tubes are inserted serially, one afteranother, into an outer tube. In another embodiment, multiple inner tubesare disposed side-by-side, so that the inner tubes provide multiplepassageways through the completed tubing. Portions of one or more of theinserted inner tubes are then melted to bond the tubes to each otherand/or to the outer tube or tubes.

In another embodiment, inner tubes are inserted within one another. Instill another embodiment, outer tubes are inserted within one another.Thus, some embodiments include more than two concentrically disposedtubes. One such embodiment is described in more detail with reference toFIG. 3.

FIG. 3 illustrates a three-dimensional angled end view of a tube 300, inaccordance with another illustrative embodiment of the invention. Thetube 300 includes an outer tube 310, an inner tube 320 (herein alsoreferred to as the intermediate tube 320) and a second inner tube 330(herein also referred to as the liner tube 330.)

The outer, intermediate, and liner tubes 310, 320 330 are eachfabricated in any desired dimensions in any suitable manner from anysuitable materials, including known fabrication methods and materials.For example, the outer tube 310 and the intermediate tube 320 optionallyhave some or all of the compositional and dimensional features,respectively, of the outer tube 210 and the inner tube 220 describedabove.

The liner tube 330 optionally is a fused-silica capillary. Theintermediate tube 320 optionally is melt bonded to the outer tube 310and/or the liner tube 330. Thus, as one example, the tube 300 has asteel outer tube 310, a thermoplastic-polymer intermediate tube 320 anda fused-silica liner tube 330. The example tube 300 provides thehigh-pressure reliability and durability of steel tubing in conjunctionwith the biocompatible properties of a fused-silica capillary forcontact with fluids passing through the tube 300.

The tube 300 also provides plumbing having a relatively narrow ID thatis well suited to nano-flow applications. Moreover, the outer tube 310supports use of narrow ID tubes 300 in conjunction with suitableconnectors, such as known connectors, that mate with relatively largediameter metallic tubing to obtain substantially fluid-tight and durableplumbing connections at pressures of up to 1,000 psi, or up to 5,000psi, or up to 10,000 psi, or greater. Some suitable connectors aredescribed below with reference to FIG. 4.

In view of the description provided herein of illustrative embodimentsfabricated from inner, intermediate and/or outer tubes, numerousalternative tubing configurations will be apparent to one havingordinary skill in the chemical separation arts. For example, someembodiments include two or more concentric outer tubes and/or two ormore concentric inner tubes. Inner and outer concentric tubes arealternated, in some embodiments, such that, for example, an inner tubeis disposed between two outer tubes and/or an outer tube is disposedbetween two inner tubes.

Returning to FIG. 1, the method 100 is useful for fabricating tubing ofa great variety of lengths. For example, tubing having a length of about1 inch or less up or a length of up to 6 feet or greater is amenable torelatively easy fabrication via the method 100. Although not required,standard lengths of commercially available tubing are amenable for usewith the method 100. A specific desired final length is obtained in someembodiments by cutting outer, intermediate, and/or inner tubes prior toinserting (Step 130) or by cutting the tubing after inserting (Step130).

The method 100 is used to fabricate both straight and curved tubing, orother desired configurations. For example, in one embodiment a length ofmetallic tubing is bent at one or more sections to provide a desiredconfiguration for use in a particular analytical instrument. An innertube is inserted (Step 130) before or after bending of the outer tube.Alternatively, an outer tube is manufactured with a non-straightconfiguration so that bending is not required.

Tubes according to many embodiments of the invention are well suited foruse with tubing connectors, such as standard connectors known to thosehaving ordinary skill in the separation arts. It should also beunderstood that the above- and below-described and illustratedconfigurations are not intended to limit application of the invention toany particular type of connector presently available or envisioned oryet to be developed. Moreover, end portions of tubes, according to someembodiments of the invention, are configured to mate with desired typesof connectors. For example, in some embodiments, an inner or outersurface of an end portion of the tube is threaded to mate with athreaded connector.

Merely as one illustrative example, convenient use of a tube with astandard connector is described with reference to FIG. 4.

FIG. 4 is a cross-sectional diagram that illustrates a portion of theplumbing of a chemical-processing apparatus, in accordance with oneembodiment of the invention. The illustrated portion is atube-and-connector assembly, which includes a tube 300 a andconventional connector components. The connector components include afitting body 410, a ferrule 420, and a fitting nut 430 (such as acompression screw.) The tube 300 a is, for example, fabricated accordingto the method 100 and/or is similar in construction to the tubes 200,300 described above. A threaded portion of the fitting nut 430 mateswith a threaded portion of the fitting body 410. The fitting nut 430,when tightened into the fitting body 410, compresses the ferrule 420against the tube 300 a to provide a seal against leaks.

Only the proximal end of the fitting body 410 is shown in FIG. 4. Thedistal end of the fitting body 410 has any desired configuration,including standard configurations. For example, the distal end may beconfigured as is the proximal end, i.e., to connect to a second tube.Alternatively, the distal end may be attached to, or an integral partof, for example, an output port of a pump, an input port of a column, ora port of another component of an apparatus. Thus, the connector isused, for example, to connect the tube 300 a to another tube of similaror different OD, to a separation column, or to another component of ananalytical instrument.

In view of the above description, one having ordinary skill in theseparation arts will understand that the tubes 300 a, 200, 300 may beused in conjunction with any suitable connectors, including knownconnectors. One suitable commercially available connector, whichincludes a fitting, ferrule, and compression screw, is the SLIPFREE®connector (available from Waters Corporation, Milford, Mass.)

In view of the description contained herein, it will be apparent to oneof ordinary skill that many other connectors are usable with varioustubing embodiments of the invention. For example, some suitableconnectors utilize a two-ferrule system. Such connectors haveapplications, for example, in high-pressure environments, for example,at pressures up to about 15,000 psi and greater.

One example of a connector that is suitable for use at very highpressure is the Swagelok gaugeable SAF 2507 super duplex tube fitting(available from the Swagelok Company, Solon, Ohio.) This connectorincludes front and back ferrules formed from different steel alloys. Theback ferrule drives the front ferrule into a fitting body and onto thesurface of a tube to create a seal.

FIG. 5 is a block diagram of a nano-flow chromatography apparatus 500,in accordance with another embodiment of the invention. The apparatus500 includes a separation column 510, a solvent reservoir 550, a solventpump 540, a sample injector 560, a detector 580, tubing 500 a connectingthe pump 540 to the reservoir 550, tubing 500 b connecting the pump tothe injector 560, tubing 500 c connecting the column 510 to the injector560, tubing 500 d connecting the column 510 to the detector 580, and acontrol module 570.

Each section of the tubing 500 b, 500 c, 500 d is similar, for example,to any of the tubing 200, 300, 300 a described above with reference toFIG. 1., FIG. 2 a, FIG. 2 b, FIG. 3 and/or FIG. 4. The tubing 500 b, 500c, 500 d has a desired inner diameter appropriate for nano-flowchromatography, for example, within a range of about 20 μm to about 40μm. Each section of the tubing 500 b, 500 c, 500 d optionally has adifferent inner diameter, as desired.

In some alternative implementations, the apparatus 500 is based on aknown high-pressure chromatographic instrument, though modified toinclude plumbing in accordance with the above described features. Onesuitable commercially available instrument is the nanoACQUITY UPLC™System (available from Waters Corporation, Milford, Mass.)

The control module 570—including, for example, a personal computer orworkstation—receives data and/or provides control signals via wiredand/or wireless communications to, for example, the pump 540, theinjector 560, and/or the detector 580. The control module 570 supports,for example, automation of sample analyses. The control module 570, invarious alternative embodiments, includes software, firmware, and/orhardware (e.g., such as an application-specific integrated circuit), andincludes, if desired, a user interface.

The column 540 contains any suitable stationary medium. For example, themedium optionally contains any suitable medium for nano-flowchromatography, such as a particulate medium known to one of ordinaryskill. Some suitable media include silica or hybrid sorbents havingparticle diameters in a range of approximately 1 μm to approximately 5μm.

In some embodiments, a particulate medium includes hybrid particles, asfound, for example, in the BEH Technology™ Acquity UPLC™ 1.7 μm columns(available from Waters Corporation, Milford, Mass.) Other embodimentsinclude larger particles, such as 3 μm or 5 μm particles. Some of theseembodiments involve trap columns.

Suitable columns are up to 25 cm in length, or greater, and have innerdiameters in a range of, for example 20 μm to 300 μm, for example, 75μm, 100 μm or 150 μm.

The pump unit 540 is configured to provide nano-flow of solvent atpressures of at least approximately 5,000 psi or 10,000 psi or greater.The pump unit includes any suitable pump components, including knownpump components, such as those found in Acquity UPLC™ liquidchromatography instruments (available from Waters Corporation, Milford,Mass.)

The nano-flow apparatus 500 is suitable for, for example, 200 nL/min to100 μL/min flow-rate separations that provide relatively goodsensitivity, resolution and reproducibility. Such separations aredesirable, for example, for biomarker discovery and for proteomicsapplications for protein identification and characterization. Thus, forexample, scientists are aided in their investigations of large proteinpopulations or proteomes to identify and quantify proteins that areeither up-regulated or down-regulated. Observed changes in proteinexpression, for example, may provide an indication of disease states.Identifying subtle changes can provide valuable information for drugdevelopment. The nano-flow separation also suitably supports subsequentmass spectrometric analysis.

As mentioned, in some embodiments of the invention, the separationcolumn itself has the above-described layered structure. Such separationcolumns have an inner diameter within a range of, for example,approximately 20 μm to approximately 300 μm, and are preferably packedwith a suitable medium, such as any of the above-described media.

Variations, modifications, and other implementations of what isdescribed herein will occur to those of ordinary skill in the artwithout departing from the spirit and the scope of the invention asclaimed. For example, though the embodiments of tubes illustrated hereinhave circular cross sections, the invention encompasses tubes that havenon-circular cross sections. Accordingly, the invention is to be definednot by the preceding illustrative description but instead by the spiritand scope of the following claims.

1. A chemical processing apparatus, comprising: a separation column; apump unit configured to deliver, to the separation column, a fluid at apressure of up to at least about 1,000 psi while at a flow rate of lessthan about 100 μL/min; at least one fluid transport tube fortransporting the fluid at least part way between the pump unit and theseparation column, the at least one fluid transport tube comprising anouter tube comprising a metallic material, a liner tube comprising fusedsilica disposed in the outer tube, and an intermediate tube comprising apolymeric material disposed between and bonded to both the outer tubeand the liner tube; and a connector comprising a ferrule and acompression screw that are disposed adjacent to an inlet or outlet endof the at least one transport tube to provide a substantiallyfluid-tight connection between the end of the at least one fluidtransport tube and an output port of the pump unit or an input port ofthe column.
 2. The apparatus of claim 1, wherein the liner tube definesa lumen having a diameter within a range of about 20 μm to about 40 μm.3. The apparatus of claim 1, wherein the connector further comprises athreaded fitting attached to the output port of the pump unit or theinput port of the column for receiving the compression screw.
 4. Theapparatus of claim 1, wherein the separation column comprises packedparticles having a diameter of less than about 2.0 μm.
 5. The apparatusof claim 4, wherein the packed particles comprise a hybrid material. 6.The apparatus of claim 1, wherein the separation column has an innerdiameter in a range of about 75 μm to about 320 μm.
 7. The apparatus ofclaim 6, wherein the separation column has a length of up to about 25cm.
 8. The apparatus of claim 1, wherein the pump unit is configured todeliver the fluid at any flow rate in a range of about 200 μL/min toabout 100 μL/min.
 9. The apparatus of claim 1, wherein the connectorprovides a substantially leak proof seal at a fluid pressure of at leastabout 10,000 psi.
 10. The apparatus of claim 1, further comprising acontrol module in data communication at least with the pump unit tocontrol processing of a sample by the apparatus.
 11. The apparatus ofclaim 1, further comprising a solvent source in fluid communication withan input port of the pump unit.
 12. The me apparatus of claim 1, whereinthe material of the outer tube comprises a material selected from thegroup of materials consisting of steel and titanium.
 13. The apparatusof claim 1, wherein a first portion of the intermediate tube has amelt-bonded fixed contact to first portions of both the outer tube andthe liner tube, wherein the fixed contact provides a fluid-tight seal toimpede fluid from reaching the unfixed contact.
 14. The device of claim13, wherein a second portion of the intermediate tube has a melt-bondedfixed contact to second portions of both the outer tube and the linertube, wherein the first and second portions of the intermediate tube aredisposed adjacent to opposite ends of the at least one transport tube.15. The device of claim 1, wherein the outer tube has an outer diameterof about 1/16 inch or less.
 16. The apparatus of claim 1, furthercomprising a mass-spectrometry unit disposed to receive an eluent fromthe separation column.
 17. The apparatus of claim 1, wherein the pumpunit is configured to deliver the fluid at a pressure of up to at leastabout 5,000 psi at the flow rate of less than about 100 μL/min.
 18. Achemical processing apparatus, comprising: a separation columncomprising an outer tube comprising a metallic material, a liner tubecomprising fused silica disposed in the outer tube, and an intermediatetube comprising a polymeric material disposed between and bonded to boththe outer tube and the liner tube; a pump unit configured to deliver, tothe separation column, a fluid at a pressure of up to at least about1,000 psi while at a flow rate of less than about 100 μL/min; a fluidtransport tube for transporting the fluid at least part way between thepump unit and the separation column; and a connector comprising aferrule and a compression screw that are disposed adjacent to an outletend of the fluid transport tube to provide a substantially fluid-tightconnection between an input port of the separation column and the outletend of the fluid transport tube.
 19. The apparatus of claim 18, whereinthe fluid transport tube comprises: an outer transport tube comprising atransport metallic material; a liner transport tube comprising fusedsilica disposed in the outer transport tube; and an intermediatetransport tube comprising a transport polymeric material disposedbetween and bonded to both the outer transport tube and the linertransport tube.
 20. The apparatus of claim 18, wherein the separationcolumn further comprises a medium having a particle size of less thanapproximately 2 μm.